Communication at the first visual synapse is mediated by L-type voltage-gated Cav1.4 Ca2+ channels. These channels are concentrated in the synaptic terminal beneath the ribbon, an organelle characteristic of synapses employing tonic neurotransmitter release. Mutation of Cav1.4 inhibits neurotransmission but can also prevent synaptic development. Loss of Cav1.4 can present as a variety of visual diseases, including congenital stationary night blindness or cone-rod dystrophy. We have validated that restoration of Cav1.4 in Cav1.4 knockout mice prior to synaptogenesis prevents these defects. But treatments for photoreceptor synaptic diseases are needed for mature individuals. In Aim 1, we will test the efficiency of synaptic rescue when Cav1.4 expression is induced in these mice after synaptogenesis is normally complete. This will be accomplished by using a strategy of tamoxifen-inducible gene expression from plasmids electroporated into mouse photoreceptors, followed by morphological and functional characterization of the synapses that form in adulthood. Regardless of when Cav1.4 expression is desired for basic or pre-clinical studies, the limited efficiency of electroporation inhibits advances in this field. Viral mediated gene expression is very effective and safe in the retina with AAV being the preferred vector. However, AAV does not have the capacity to express large genes like Cav1.4. In Aim 2, we will develop multiple AAV vectors expressing fragments of Cav1.4 engineered for post-translational recombination by trans-intein splicing and test the effectiveness of this strategy for achieving expression of Cav1.4 in adult mouse photoreceptors. Success in this area will not only accelerate research on Cav1.4 but could be adapted to other large genes that are prime candidates for retinal gene therapy but currently cannot be accommodated in AAV. Together, this work exploring the regenerative capacity of photoreceptor synapses and expansion of the utility of AAV for retinal gene therapy, will greatly advance efforts to save and restore vision.